Tracer‐Based Metabolic NMR‐Based Flux Analysis in a Leukaemia Cell Line

Abstract

High levels of reactive oxygen species (ROS) have a profound impact on acute myeloid leukaemia cells and can be used to specifically target these cells with novel therapies. We have previously shown how the combination of two redeployed drugs, the contraceptive steroid medroxyprogesterone and the lipid‐regulating drug bezafibrate exert anti‐leukaemic effects by producing ROS. Here we report a 13C‐tracer‐based NMR metabolic study to understand how these drugs work in K562 leukaemia cells. Our study shows that [1,2‐13C]glucose is incorporated into ribose sugars, indicating activity in oxidative and non‐oxidative pentose phosphate pathways alongside lactate production. There is little label incorporation into the tricarboxylic acid cycle from glucose, but much greater incorporation arises from the use of [3‐13C]glutamine. The combined medroxyprogesterone and bezafibrate treatment decreases label incorporation from both glucose and glutamine into α‐ketoglutarate and increased that for succinate, which is consistent with ROS‐mediated conversion of α‐ketoglutarate to succinate. Most interestingly, this combined treatment drastically reduced the production of several pyrimidine synthesis intermediates.

Positional Enrichment by Proton Analysis (PEPA): A One‐Dimensional 1H‐NMR Approach for 13C Stable Isotope Tracer Studies in Metabolomics

“…In comparison with 13 C-NMR, TOCSY and HSQC,P EPA improves sensitivity,accelerates the elucidation of 13 Cpositions in labeled metabolites and the quantification of the percentage of stable isotope enrichment. Altogether,P EPAp rovides an ovel framework for extending the high-throughput of 1 H-NMR metabolic profiling to stable isotope tracing in metabolomics,f acilitating and complementing the information derived from 2D-NMR experiments and expanding the range of isotopically enriched metabolites detected in cellular extracts.…”

“…Elucidating the flow of chemical moieties through the complex set of metabolic reactions that happen in the cell is essential for understanding the regulation of metabolic pathways. [2] Fort hat, stable isotope tracer studies are needed, which usually require model cell lines that are fed as table isotopically labeled substrate.D ue to the unique ability of NMR to characterize isotopomers of metabolites by detecting labeling patterns in individual atoms, 13 C-NMR in combination with selective 13 Cstable isotope tracers have traditionally been used to analyze 13 Cenrichment in isolated metabolites [3] and biological crude mixtures, [4][5][6] enabling models of metabolic flux to be generated. [7] Thea dvent of comprehensive metabolic profiling technologies has broadened the coverage of these studies allowing for an unbiased mapping of fluxes through multiple metabolic pathways.…”

“…PEPA detects the position of carbon label in isotopically enriched metabolites and quantifies their fractional enrichment in 1D-1 H-NMR spectra. In principle,t he ability of 1D-1 H-NMR to observe 13 C-satellite peaks makes it suitable for tracer studies. [3] However,d irect quantification of 13 C-satellites in 1D-1 H-NMR spectra of cell extracts is generally only possible for af ew metabolites characterized by well-resolved resonances with recognizable 13 C-satellite peaks such as lactate and alanine.…”

“…U2OS cells were subjected during 6hours to culture medium containing 13 C-enriched substrates (either uniformly labeled glucose [U-…”

“…In order to confirm PEPAsr esults, 13 C-NMR and 2D-edited NMR experiments (TOCSY,HSQC and HMBC) were acquired on representative sample extracts grown either in unlabeled or labeled substrates. (Figure 2A).…”